Vascular endothelial cells (ECs), located at the interface between the blood and the vessel wall, are exposed to the mechanical environment resulting from hemodynamic activities. Fluid shear stress is the hemodynamic force acting tangentially on the vascular ECs and it plays significant roles in atherogenesis and reperfusion injury. Many genes encoding for growth factors (e.g., platelet derived growth factor and transforming growth factor xcex2-1), vasoconstrictors (e.g., endothelin-1), vasodilators (e.g., nitric oxide synthase), adhesion molecules (e.g., intercellular adhesion molecule-1, ICAM-1), and monocyte chemoattractants (e.g., monocyte chemotactic protein-1, MCP-I) in the ECs are modulated by fluid shearing (see7 for review). The induction of some, and perhaps the majority, of these inflammation-related genes is rapid and transient, and de novo protein synthesis is not required. These are the characteristics of the expression of immediate early (IE) genes induced by mitotic factors and agonists. The phorbol ester 12-0-tetradecanoyl-13-phorbol-acetate (TPA) responsive element, TRE, mediates the expression of many IE genes through its interaction with the transcription factor AP-1, a Jun/Fos heterodimer or a Jun/Jun homodimer (1). A divergent TRE in the 5xe2x80x2 promoter region of the MCP-1 gene has been found by the present inventors to be responsible for its mechanical inducibility (41). Consensus TRE with the sequence TGACTACA is sufficient for the shear-induced reporter activities in different types of cells. The applied fluid shearing probably exerts its actions on the cellular membrane to initiate biochemical signals which can then be transduced through the cytoplasm into the nucleus where the activation of AP-I/TRE occurs.
A major process through which extracellular stimuli can be transmitted into cells involves the membrane-associated p21ras and its downstream cytoplasmic kinase pathways, especially the members in the mitogen-activated protein kinases (MAPK) family. p21ras is a small GTPase molecule that plays a key role in the signal transduction pathways of cellular responses to stimuli by mitogins, cytokines, environmental stresses, and UV irradiation. p21ras cycles between an active GTP-bound state and an inactive GDP-bound state, thereby functioning as a molecular switch in response to extracellular stimuli in the control of normal and transformed cell growth. Activated p21ras triggers two protein kinases, Raf-1 and MEK (MAPK kinase) kinase (MEKK) which activate the downstream MAPKs, including c-Jun NH2-terminal kinases (JNK) and extracellular signal-regulated kinases ERK (11,32). Raf-1 activates ERK but not JNK, whereas MEKK mediates preferentially JNK over ERK (32,48). In different types of cells in response to UV irradiation, Ha-Ras expression, and osmotic shock, JNK kinase (JNKK) activates JNK by phosphorylating the Thr-Pro-Tyr phosphorylation sites, and the activated JNIK binds to c-Jun to specifically phosphorylate the -63 and -73 amino acids at the N-terminal (10, 29). In response to Ha-Ras expression, serum growth factor, or phorbol ester TPA stimulation, MEK activates ERK which in turn phosphorylates the transcription factor p62 ternary complex factor (p62TCF), leading to the activation of c-Fos (5, 18, 30, 37). In R.EF-52 fibroblasts, the activation of AP-I/TRE by these stimuli is mediated through ERK (16). It is not known where and how mechanical stimuli are transduced to biochemical signals. p21ras is a membrane-associated protein and its activation of the downstream Raf-1 and MEKK is through direct interactions on the membrane (27, 28, 36, 38, 43). Wang et al. (47) suggest that the integrins on the basal membrane constitute a mechano-receptor and that stress fibers are necessary to transmit the applied forces. Similarly, Davies et al. (8) suggest that focal adhesion complex at the abluminal endothelial membrane are mechanically responsive elements coupled to the cytoskeleton.
Ras can activate both ERK and JNK pathways (27, 32). The signaling in response to growth factors such as epidermal growth factor (EGF) and nerve growth factor (NGF) is mediated through both the Ras/ERK and Ras/JNK pathways in PCI2, MRCS, and HeLa cells (32, 33). In contrast, inflammationxe2x80x94related cytokines (e.g., TNF and IL-1), environment stresses (e.g., osmoic pressure), and UV irradiation selectively activate the Ras/JNK, but not the ERK, pathway (17, 19, 26, 32, 42, 44). Mechanical shearing is a form of force borne by vascular ECs and many other cell types, such as the osteoblasts, under physiological conditions. The application of such physiological forces on static cells cultured in the flow chambers provides a sudden change of hemodynamic environment. This in vitro system mimics the pathophysiological changes during reperfusion after flow stoppage. Fluid shearing of vascular EC caused the activation of JNK by more than 10-fold and the activation of ERK by a much lesser magnitude (1.8 fold) and shorter duration (FIG. 3). Morooka et al. demonstrated that reperfusion of ischemic kidney induced a rapid activation of JNK (35). Bogoyevitch et al. reported that reperfusion of rat heart induced a 10- to 50-fold activation of JNK, but not ERK (2).
While several laboratories have shown that fluid shearing induces a variety of transient responses in the endothelial cytoplasm and nucleus, there is little, if any, knowledge on how ECs transduce the mechanical stimuli into biochemical signals which ultimately activate the downstream gene expression. The present inventors have found that fluid shearing, a physiological form of hemodynamic forces, activates p21ras in ECs in a rapid and transient manner, and that it is followed by the activation of the MEKK-JNK pathway, leading to the induction of the AP-1/TRE mediated gene expression in the nucleus. In contrast, the ERK pathway is weakly activated by fluid shearing and is not essential for the shear-induced activation of AP-I/TRE. These results indicate that hemodynamic forces share the same signaling pathways as a variety of stimuli, including osmotic pressure, chemical stress, and UV irradiation, in activating the promoter regions of IE genes.
Percutaneous transluminal coronary angioplasty (PTCA) causes restenosis mainly from proliferation of vascular smooth muscle cells (VSMC) gene expression and proliferation. Recognizing this, an effective gene therapy method for the reduction of the high incidence of restenosis after angioplasty is provided in the present invention.
The present invention demonstrates that p21ras plays critical roles in the responses of vascular ECs to fluid shearing. First, the guanine nucleotide exchange on Ras, i.e., the conversion of Ras-GDP to Ras-GTP, was promoted by fluid shearing. Second, the dominant negative mutant of p21ras RasN17, inhibited the shear-induced signal transduction pathway including JNK and its downstream c-Jun transcriptional activity. Third, RasNI7 also abrogated the reporter activities of 4xc3x97TRE-Luc and MCP1-Luc-540, two chimeric constructs whose induction by fluid shearing is mediated by AP-1/TRE (41). The present inventors establish that the mechanical-biochemical transmitting process occurs at least in part on the cellular membrane. The present inventors propose that the signals initiated by fluid shearing originate from the abluminal side of ECs. In contrast, the burst production of nitric oxide is dependent upon the activation of G proteins (25) which are located at the luminal surface.
Besides the activation of ERK, the inventors also demonstrate that the blockage of the p21ras-RAF-ERK pathway by Raf.301, K7 I R, or K52R did not affect the TRE-mediated reporter activities in response to fluid shearing (FIG. 6). In contrast, the blockade of the p21ras-MEKK-JNK pathway by either MEKK(K-M) or JNK(K-R) significantly attenuated the shear induced reporter activities (FIG. 5). MEKK(K.M) and JN.K(K-R), which were mutated at the ATP binding sites with the conserved Lys replaced by either a Met or an Arg, act like dominant negative mutants of .MEKK and JNK, respectively (32; unpublished results of B. Su). Thus, the induction of MEKK-JNK by fluid shearing plays a key role for the activation of the downstream AP1/TRE, an effect which is probably mediated through c-Jun. On the other hand, the activation of cFos, which is dependent on ERK/TCF (9), seems to be not necessary for such an activation. It appears that the Jun-Jun homodimer, rather than the Jun-Fos heterodimer, serves as the activator in this shear-elicited signal transduction pathway. Presumably, the shear-induced activation of JNK by JNKK phosphorylates the pre-existing, latent c-Jun in the cytoplasm, and this is followed by the translocation of the activated c-Jun into the nucleus where the Jun-Jun homodimer activates the target gene by interacting with TRE. Transactivation assays using RSV-Jun, an expression plasmid encoding c-Jun, showed similar effects as fluid shearing, indicating the MCP1-Luc-540 is activated by JNK (41). These results, taken together, suggest that the p21ras .MEKK-JNKK-JNK pathway is necessary and sufficient to activate the AP-1/TRE-mediated gene expression in ECs in response to fluid shearing (FIG. 8). In addition to the phosphorylation of the pre-existing c-Jun, the induction of AP-1 activity may also occur at the transcriptional level. Mechanical shearing induces c-jun mRNA and it remains at an elevated plateau level for at least 4 hr (21). It is not known whether the activated JNK in the sheared cells would activate the de novo synthesized c-Jun through the phosphorylation of Ser-63 and -73 (33), and if it does, what would be duration over which the activated c-Jun homodimer can activate the downstream genes.
The duration of MAPK activation by different extracellular stimuli may determine whether the cells can elicit differentiation or proliferation responses (31). In PCI2 cells, ERK activation is sustained for several hours following NGF stimulation, thus leading to differentiation of these cells to become sympathetic neurons. In contrast, the response is transient after EGF stimulation, and the result is proliferation rather than differentiation (45). Whether the activation is sustained or transient is dependent on the receptor tyrosine kinases (RTKs) which activate p21ras. The cellular responses to fluid shearing, including the activation of p21ras, JNK and the downstream IE genes (e.g., the MCP-1 gene), are all transient and rapid. The cells are conceivably desensitized by the applied mechanical force following the initial activation.
The present inventors have found that pre-shearing desensitizes ECs against further TPA-induced ERK phosphorylation (41). Such mechanically induced transient responses and desensitization have their physiological significance in vascular ECs. Serving as the barrier between blood and vessel wall, these cells need a desensitization mechanism to protect them from the continuous stimulation imposed by the hemodynamic forces. In bends and bifurcations where the shearing forces are low and the blood flow is disturbed, there may not have been the same degree of desensitization as that in the lesion resistant areas where the endothelium is subjected to a relatively constant laminar flow with high shearing forces. When cumulated over years, the small differences in the mechanical environment (i-e., the magnitude of shearing forces and flow pattern) between the cells in these different regions may have considerable pathophysiological consequences.
It is intriguing that Sos can be upstream to the fluid shearing-activated p21ras. The present inventors have found that the negative mutants of GTb2 and Sos can also partially block the shear-induced 4xc3x97TRE-P1 luc and MCPI-luc-540 in BAEC (23). Grb2 is an adapter protein which contains one src homology domain 2 (SH2) and two SH3 domains. GTb2 binds to Sos, a guanine nucleotide exchange factor specific to p21ras. Thus, the upstream mechanisms by which the mechanobiochemical transduction activates Ras pathway may be similar to those for growth factor stimuli. It remains to be investigated how common upstream signals diverge to activate JNK (mechanical stimuli) and ERK(growth factor).
The p21ras-MEKK-JNK-AP-1/TRE pathway provides a molecular mechanism for the signal transduction in endothelial responses to mechanical stimulation. RTKs may be the xe2x80x9cmechanical force receptors/sensorsxe2x80x9d on the membrane that execute the mechano-biochemical transduction to activate such a pathway. The similarity between fluid shearing and EGF in inducing endothelial responses including the involvement of SH2-containing molecules such as Grb2 suggests that RTK, especially EGF receptor subfamily, may play an important role to transduce the mechanical stimuli into biochemical signals. Recently, it has been shown that the small GTP-binding proteins Rac and Cdc42 are upstream to JNK (6, 34). Constitutively activated Rac and Cdc42 stimulate the catalytic activity of JNK (34). Dominant negative mutants of Rac and Cdc42 effectively reduce the EGF-activated JNK (6). Thus, Rac and Cdc42 are crucial intermediates in the signaling pathway leading from activated RTKs to JNK. These findings reinforce the hypothesis that RTKs are candidates for the mechanical force sensor, but direct experimental evidence is still lacking. Whatever the sensor mechanism, the p21ras-MEKK-JNK-AP-1/TRE pathway seems to be part of a coordinated programming, including other possible components such as G proteins, Ca2+, integrins, etc. The synergism and/or cross communication among these different signaling pathways probably plays significant roles in constituting the endothelial responses to hemodynamic forces.
The present invention in one aspect provides for a method of inhibiting or reducing tissue injury attendant to angioplasty through the introduction of a Ras therapeutic gene capable of blocking a Ras signal transduction pathway. In some embodiments, the method comprises introducing a Ras therapeutic gene capable of blocking a Ras signal transduction pathway. By way of example, and not exclusion, the Ras signal transduction pathway that is to be blocked as part of the method is selected from the group consisting of an MEKK pathway, a JNKK pathway, an ERK pathway, and an MEK pathway. These pathways are among those contemplated by the present inventors to be useful in blocking Ras expression.
In particular embodiments, the Ras therapeutic gene is more particularly described as a Ras mutant gene, and even more particularly as a dominant negative mutant Ras gene. In some embodiments, the method contemplates the delivery of this gene and of the Ras therapeutic gene more generally, in adenovirus. However, it is contemplated that any variety of carrier vehicles may be used in the delivery of the gene, including via plasmid, retrovirus, adenovirus or any other molecule capable of effectively providing the therapeutic gene or a portion thereof to a population of cells targeted for treatment.
Methods for introducing foreign genetic material into a cell are well known to those of skill in the art, and said standard protocols for introducing a gene of interest into a cell are contemplated by the present inventors as within the scope of the present invention.
In another aspect, the invention provides a method for reducing restenosis employing the Ras therapeutic gene described herein, or a therapeutically functional fragment thereof. In some embodiments, the method comprises administering to a patient at risk of restenosis a therapeutic Ras gene, particularly within an adenovirus. Such adenovirus is preferably replication defective, and will include a substituted E1 region. In some embodiments, the adenoviral construct will comprise a Ras N17 adenovirus package, wherein the Ras N17 adenovirus package is a non-replicating adenovirus genome containing a Ras N17 expression cassette.
In yet another aspect, the invention provides a therapeutic composition comprising a replication deficient recombinant adenovirus construct comprising a therapeutic Ras gene as described herein.
In other potential applications, the prevention and treatment of restenosis in other conditions, e.g., arterio-venous grafts used in hemodialysis and intra-hepatic portal-caval shunts used in portal hypertension. Other possible applications are for the inhibition of smooth muscle proliferation in smooth muscle tumors of the uterus and other organs.
As used in the description of the present invention, the term xe2x80x9caxe2x80x9d is intended to mean one or more.
Other applications and uses of the present invention not specifically articulated herein are intended as within the scope of the present invention.